The overall goal of this project is to understand the structural basis of CLCA1 activation and its role in airway biology in health and disease, in order to translate this knowledge into treatments for asthma and COPD, a current unmet need. CLCA1 is a potent modulator of calcium-activated chloride channels (CaCCs) and also a central mediator in mucous cell metaplasia, the process that leads to mucus overproduction. In this project we will investigate the structural and biochemical basis of CLCA activation of CaCCs, and investigate the role that CLCA1-mediated channel activation plays in mucous cell metaplasia. In our preliminary results, we demonstrate that CLCA proteins contain a consensus cleavage site that is recognized by a unique zinicin metalloprotease domain located within the N-terminus of CLCA itself. Furthermore, we show that this self- cleavage is required for hCLCA1 to activate CaCCs. These data suggest that CLCA1 is synthesized in a full- length inactive form and that self-cleavage is required to produce an active form of the protein. This project will focus on the structural and biochemical analysis of regulation of the metalloprotease domain activity, since it is necessary to produce the active form. We will then characterize the structura changes that occur upon activation by determining the structures of the full-length inactive and active forms of CLCA1. Finally, we will address the functional role of CLCA1 features in CaCC activation and the role of the channel in mucous cell metaplasia. Understanding how CLCA1 activity is regulated by its own metalloprotease domain and the downstream functional consequences of this regulation will facilitate the design of CLCA1 inhibitors for anti- mucus therapeutics.